Upload
others
View
14
Download
6
Embed Size (px)
Citation preview
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17
http://www.sciencepublishinggroup.com/j/ijbbmb
doi: 10.11648/j.ijbbmb.20200501.12
ISSN: 2575-5889 (Print); ISSN: 2575-5862 (Online)
Determination of Caffeine in Coffee Samples by High Performance Liquid Chromatography and Ultra Violet - Visible Spectrophotometry Methods from Wollega, Ethiopia
Shibiru Eticha, Tesfa Bedassa
Department of Chemistry, Wollega University, Nekemte, Ethiopia
Email address:
To cite this article: Shibiru Eticha, Tesfa Bedassa. Determination of Caffeine in Coffee Samples by High Performance Liquid Chromatography and Ultra Violet -
Visible Spectrophotometry Methods from Wollega, Ethiopia. International Journal of Biochemistry, Biophysics & Molecular Biology.
Vol. 5, No. 1, 2020, pp. 8-17. doi: 10.11648/j.ijbbmb.20200501.12
Received: May 15, 2020; Accepted: June 1, 2020; Published: August 27, 2020
Abstract: In this research caffeine content in coffee sample from Abe Dongoro, Sasiga, Gida Ayana and Sibu Sire of
Wollega administrative zone of Ethiopia were determined using High Performance Liquid Chromatography (HPLC) and UV-
Vis Spectrophotometry methods. Caffeine in aqueous extract of coffee samples was extracted with dichloromethane prior to
analysis by UV-Vis spectrophotometry method and dichloromethane was evaporated from the extract and the extract was
dissolved in water (HPLC grade) to determine caffeine contents in coffee samples using HPLC method. The linearity of the
HPLC and UV-Vis spectrophotometry methods were R2 = 0.9999 and R
2 = 0.9997 respectively. HPLC and UV-Vis
spectrophotometry methods were found to be accurate with recoveries of 97.5% and 117.4%, respectively. Limits of detection
(LOD) obtained were 0.148 mg/L for HPLC method and 0.284 mg/L for UV-Vis spectrophotometric method. The caffeine
contents in coffee samples obtained using UV-Vis spectrophotometry method was 3.42, 2.638, 2.207 and 2.986 mg/L for Abe
Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples respectively. Similarly, using HPLC method the caffeine contents
in coffee samples obtained was 1.871, 1.601, 1.307, 1.83 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee
samples. There is a significant difference between the caffeine contents in coffee samples obtained by the two methods.
Keywords: Coffee Samples, Caffeine, UV-Vis Spectrophotometry, High Performance Liquid Chromatography,
Horro Guduru Wollega, East Wollega
1. Introduction
Coffee is one of the popular beverages that is widely used
around the world due to its pleasant flavor, aroma and fitness
benefits. Botanically it belongs to genus Coffea in the
Rubiaceae family. Genus Coffea contains over 80 diverse
numbers of species. However, Coffea arabica and Coffea
robusta are the main genus Coffea species [1]. Arabica coffee
is grown-up in about tropical and sub-tropical of 80
countries. Ethiopia is among these countries, which heavily
depend on coffee exports for foreign exchange earnings [2].
Coffee has a complex mixture of chemical constituents
such as phenolic compounds, caffeine, trigonelline,
chlorogenic acids, protein, lipids and minerals [3-5]. The
chemical content of coffee varies with species, geographical
source, climatic parameters, cultivation system (organic
versus conventional system) [6].
Caffeine is a natural alkaloid widely used in food industry
as a psycho stimulant in beverages and foods for information
processing and cognitive performance [7]. It is found in tea
[8], chocolate [9], caffienated, decaffienated beverages and
energy drinks [10], Arabica coffee [11] and Robusta coffee
[12].
Caffeine is used to increases alertness, improves short-
term memory and increases the efficiency of certain drugs.
However, over dose use of caffeine is a matter of health
concerns such as gastric acid secretion and higher risk of
miscarriage [13]. Therefore, monitoring of caffeine content in
food and beverages is an important challenge of food quality
control.
Various analytical and electro analytical techniques such as
high performance liquid chromatography [14, 15], Fourier
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17 9
transforms near infrared reflectance spectroscopy [16], UV-
Vis spectrophotometery [17, 18] and voltammetry [19] are
reported for the determination of caffeine contents in food
and beverages.
Caffeine contents in coffee samples grown at different part
of Ethiopia were studied using several analytical techniques.
However, to this date, there is no published research finding
that made use of Ultra Violet-Visible Spectrophotometery
(UV-Vis) and High Performance Liquid Chromatography
(HPLC) for the determination of the caffeine contents in
coffee samples from Eastern and Horro Guduru Wollega
Zones in the Western part of Ethiopia. Therefore, the
objective of this study was to determine caffeine in coffee
samples from Abe-Dongoro, Sasiga, Gida-Ayana and Sibu-
Sire districts in Wollega zones of Western Ethiopia by High
Performance Liquid Chromatography (HPLC) and Ultra
Violet-Visible Spectrophotometery (UV-Vis) methods.
2. Experimental
2.1. Study Area
Coffee bean samples were collected from three selected
districts of Eastern Wollega administrative zone from the
Sasiga, Gida-Ayana, Sibu-Sire and Abe-Dongoro district of
Horro- Guduru Wollega administrative zone directly from
the farmers’ farmlands. The geographical maps of the
sampling sites were displayed in Figure 1. The districts were
preferred purposely as they are major coffee producing parts
of the administrative zone.
2.2. Chemicals and Reagents
Chemicals and reagents used were of analytical grade;
standard caffeine powder (Aldrich, Germany), HPLC grade
methanol (Aldrich, Germany), HPLC grade water and
dichloromethane (Aldrich, Germany).
Figure 1. Geographical location of sampling site.
10 Shibiru Eticha and Tesfa Bedassa: Determination of Caffeine in Coffee Samples by High Performance Liquid
Chromatography and Ultra Violet - Visible Spectrophotometry Methods from Wollega, Ethiopia
2.3. Instruments
Caffeine contents in coffee samples was determined using
UV-Vis spectrophotometry (18- 1884-01-0076, spectral
bandwidth 2.00 nm) and HPLC system (Agilent1260,
Germany) equipped with a pump (G1310B), column
(G1316A/ Agilent poroshell-C18 (4.6 x 250 mm, 2.7µm)),
auto sampler (G1329B), variable wavelength detector
(G4286B) and Chemistation software.
2.4. Preparation of Standard Solutions
For analysis by HPLC, the stock solution of standard
caffeine (1000 mg/L) was prepared by dissolving 100 mg of
standard caffeine in 50 mL warm, ultrapure water in 100 mL
of volumetric flask and filled to the final volume with
ultrapure water. Intermediate standard solution of caffeine
(100 mg/L) was prepared from the stock solution. Finally,
concentrations of 2, 4, 8 and 16 mg/L caffeine solution were
used to construct calibration curve.
For the UV-Vis spectrophotometric analysis, the stock
solution of standard caffeine (1000 mg/L) was prepared by
dissolving 100 mg of standard caffeine in 50 mL
dichloromethane in 100 mL of volumetric flask and filled to
the final volume with dichloromethane. Intermediate standard
solution of caffeine (100 mg/L) was prepared by diluting the
stock solution with dichloromethane. Finally concentrations of
0, 2, 4, 8 and 16 mg/L were prepared by diluting the working
standard for calibration. For the quantitative determination of
caffeine contents in coffee samples the λmax (272 nm) of
caffeine standard was selected from the reported literature, this
is because of the characteristics observed peak of caffeine at
271–276 nm [20, 21].
2.5. Coffee Sample Preparation
A 20 g of coffee bean from each sample was roasted by
using the conventional coffee roasting machine. After
roasting, the coffee bean samples were cooled at room
temperature. Each of the roasted and cooled coffee bean
samples was grounded and homogenized using an electric
coffee grinder machine. After that, each of the coffee powder
was screened through 300 µm sieve to get a uniform mixture
and kept in plastic bag at room temperature until it is used for
the extraction. The extraction of caffeine from the aqueous
solution into dichloromethane was carried out by the reported
methods [22, 23].
Trigonelline, chlorogenic and caffeic acids are the primary
interference in the quantitative determination of caffeine in
coffee samples using UV-Vis spectrophotometry method. In
order to overcome this difficulty the coffee samples was first
dissolved in water and then the caffeine was extracted from
the aqueous extract using dichloromethane. The efficiency of
dichloromethane to extract caffeine from coffee beans is 98–
99% [23]. For HPLC analysis, 2 mL aliquot from
dichloromethane extract was pipetted into test tube and the
dichloromethane was evaporated; the residue was dissolved
in 2 mL HPLC grade water.
2.6. Chromatographic Analysis
The samples and standard solutions of 20 µL were injected
into the column with a flow rate of 0.8 mL/min using a
mobile phase consisting of water and methanol (75:25, V/V).
The column temperature was kept at 30 OC and data rate at
10 Hz. Chromatographic data for caffeine were collected at
272 nm.
2.7. Method Validation
Linearity of the calibration curves was evaluated based on
the magnitude of the regression coefficient (R2). Accuracy
was validated by using spike-recovery method. The limit of
detection (LOD) and limit of quantification (LOQ) of the
analytical method is obtained based on the standard deviation
of response and slope of the calibration curve [24]. In this
study, LOD and LOQ of the methods were calculated as
LOD=3.3 σ/s; LOQ=10 σ/s, were, s is the slope of the
calibration curve and σ is the standard deviation of the y-
intercept of the regression line. Differences between groups
were assessed by one-way analysis of variance (ANOVA).
The results with P < 0.05 were regarded to be statistically
significant.
3. Result and Discussion
3.1. Analytical Performance Characteristics
The performance parameters for both HPLC and UV-Vis
spectroscopic analysis are presented in Table 1. The
calibration curve for both of the analysis methods showed an
excellent linear fit. LOD for HPLC and UV-Vis methods
were 0.148 and 0.284 mg/L, respectively. Recoveries of both
methods were satisfactory. Thus, the proposed methods were
appropriate for determination of caffeine contents in coffee
samples.
Table 1. Performance parameters of the HPLC and UV-Vis
spectrophotometry methods.
No Parameters HPLC method UV-Vis method
1 Coefficient of correlation 0.9999 0.9997
2 LOD (mg/L) 0.148 0.284
3 LOQ (mg/L) 0.449 0.86
4 Recovery 97.5% 117.4%
3.2. Quantity of Caffeine in Coffee Samples
The identity of the analyte was determined by comparing
the retention time extracted from the coffee samples with
retention time of standard caffeine. Under the optimized
experimental condition, the retention time of caffeine
obtained was 10.6 minutes. In addition, the identity of
caffeine was confirmed by spiking one of the coffee extract
with standard caffeine. An increase in peak area after caffeine
standard addition to the coffee extract supported the
identification of the caffeine extracted from the samples. The
extracts of the coffee samples were analyzed using HPLC
method to determine the caffeine contents in the samples
(Table 2).
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17 11
Table 2. Caffeine content in coffee samples obtained using HPLC method.
Coffee samples Retention time Peak area Concentration (mg/L) % of Caffeine (w/w)
Abe Dongoro 10.649 179.856 1.871 1.12
Gida Ayana 10.627 156.336 1.601 0.96
Sasiga 10.637 130.671 1.307 0.78
Sibu Sire 10.637 176.324 1.830 1.1
Sasiga (duplicated) 10.616 130.157 1.301 0.78
Gida Ayana (4000 mg spiked) 10.490 212.578 2.246 1.35
The caffeine content in coffee samples obtained using
HPLC method was in the range of 0.78- 1.12% (w/w). The
highest caffeine content was obtained in the Abe Dongoro
coffee sample which gave a concentration of 1.871 mg/L or
1.12% (w/w), followed by a Sibu Sire coffee sample which
gave a concentration of 1.830 mg/L or 1.10% (w/w). The
least caffeine content was obtained in Sasiga coffee sample
which gave a concentration of 1.307 mg/L or 0.78% (w/w)
(Table 2). The results of the present study are comparable
with the results reported in the literature 0.06-2.55% (w/w)
[25], 0.84-1.15% (w/w) [26] and 0.8-1.4% (w/w) [27] for
caffeine in coffee samples.
The absorbance of the caffeine in each coffee sample
solution was measured using UV-Vis spectrophotometry at
272 nm against the corresponding blank (dichloromethane).
Caffeine content in coffee samples obtained by UV-Vis
spectrophotometry method was presented in Table 3.
Table 3. Caffeine content in coffee samples obtained by UV-Vis spectrophotometry method.
Coffee samples Conc.(mg/L) (mean ± sd) % of Caf (w/w) (mean ± sd)
Abe Dongoro 3.420±0.059 2.052±0.036
Gida Ayana 2.638±0.026 1.583±0.015
Sasiga 2.207±0.009 1.322±0.005
Sibu Sire 2.986±0.016 1.792±0.01
Gida Ayana (4000 mg spiked) 3.420± 0.076 2.052±0.046
The highest caffeine content was obtained in the Abe
Dongoro coffee sample which gave a concentration of 3.420
mg/L or 2.052% (w/w), followed by a Sibu Sire coffee
sample which gave a concentration of 2.986 mg/L or 1.792%
(w/w). The least caffeine content was obtained in Sasiga
coffee sample which gave a concentration of 2.207 mg/L or
1.322% (w/w).
The caffeine content in coffee samples obtained using UV-
Vis spectrophotometry method was in the range of 1.322 -
2.052% (w/w). There was a significant difference (p < 0.05)
in caffeine contents among all the coffee samples. The results
of the present study are comparable with the caffeine
contents reported in the literature 0.9-3.01% (w/w) [21],
1.24-2.54% (w/w) [28] for caffeine in coffee samples.
The caffeine content in coffee samples obtained using UV-
Vis spectrophotometry method was compared with the
caffeine content in coffee samples obtained using HPLC
method (Table 4). In contrast to this, the UV-Vis
spectrophotometry method is much faster than the HPLC
method. The caffeine contents in coffee samples were found
in the range of 1.322 - 2.052% (w/w) using UV-Vis
spectrophotometry method and 0.78 - 1.12% (w/w) using
HPLC method.
Table 4. Comparison of caffeine content in coffee samples% (w/w) obtained using UV-Vis spectrophotometry and HPLC methods.
No. Sample name Method
UV-Vis HPLC
1 Abe Dongoro 2.052 1.12
2 Gida Ayana 1.583 0.96
3 Sasiga 1.322 0.78
4 Sibu Sire 1.792 1.1
The present results indicates that the caffeine contents in
coffee samples obtained using UV-Vis spectrophotometry
method was higher than the caffeine contents in coffee
samples obtained using HPLC method.
Application of statistical analysis (paired t-test at 0.05
levels) to the data obtained by the two methods indicated that
there is significant difference between them. The caffeine
contents of coffee samples obtained in this study was
compared with caffeine values reported for coffee samples
from different parts of the Ethiopia (Table 5).
Table 5. Comparison of results of the present study with the reported literature
Method % Caf (w/w) Origin of coffee References
UV-Vis 0.97 - 1.53 (n=4) North West Ethiopia Belete Tewabe & Solomon Libsu, 2015 [29]
UV-Vis 0.6 - 0.9 (n=16) Hararghe Ephrem Demissie et al., 2016 [30]
HPLC 0.6 - 1.1 (n=9) South West Ethiopia Mulu Hagos et al., 2018 [22]
HPLC 0.62 - 1.99 (n=5) Gojjam Maria et al., 2000 [31]
12 Shibiru Eticha and Tesfa Bedassa: Determination of Caffeine in Coffee Samples by High Performance Liquid
Chromatography and Ultra Violet - Visible Spectrophotometry Methods from Wollega, Ethiopia
Method % Caf (w/w) Origin of coffee References
HPLC 1.31-1.36 (n=6) Bale Legesse Adane et al., 2018 [32]
UV-Vis 1.32 - 2.052 (n=4) *Wollega Present work
HPLC 0.78 - 1.12 (n=4) *Wollega Present work
* Wollega = Eastern and Horro Guduru.
The caffeine content in coffee samples reported in the
present study using UV-Vis spectrophotometry method is
slightly greater than the reported caffeine contents in North
West Ethiopia and Hararghe coffee samples using UV-Vis
spectrophotometry method and slightly greater than the
reported caffeine contents of South West Ethiopia, Gojjam
and Bale coffee samples using HPLC method.
The obtained caffeine content in coffee samples in this
study using HPLC method is slightly smaller than caffeine
contents in Gojjam coffee samples and comparable with the
South West Ethiopia coffee samples reported by HPLC
method and somewhat smaller than the caffeine contents in
North West Ethiopia coffee samples reported using UV-Vis
spectrophotometry method.
In general the caffeine contents in coffee samples
obtained in the present work is in the range of caffeine
contents of export standard Ethiopian coffee samples (0.46-
2.82% w/w) reported using HPLC method [31]. The
difference in caffeine contents in coffee samples are due to
several factors such as coffee variety, genetic properties of
the cultivars, maturity of the beans at harvest, harvesting
method and postharvest processing conditions (fermentation,
washing, drying, storage), agricultural practices (shade,
pruning, fertilization), environmental factors (soil, altitude,
sun exposure), climatic parameters (rainfall, temperature),
method of preparation (the brewing of coffee) and
analytical methods used for the determination of caffeine in
coffee samples [6, 33].
4. Conclusion and Recommendations
In this study, HPLC and UV-Vis spectrophotometry
methods were successfully applied for the determination of
caffeine contents in coffee samples grown in Wollega from
Sasiga, Gida Ayana, Sibu Sire and Abe Dongoro woredas.
Variations of the caffeine contents in coffee samples were
observed; these variations depend on the geographical
origin of the coffee samples and the analytical methods
used for caffeine content determination in coffee samples.
Significant difference was observed between the caffeine
value obtained by HPLC and UV-Vis spectrophotometry
methods. The caffeine content in coffee samples obtained in
this study is in the range of caffeine contents of export
standard Ethiopian coffee samples. Further study is required
in which coffee samples could be analyzed with statistically
sufficient data, coffee variety, agricultural and
environmental factors as well as other components of coffee
beans such as chlorogenic acid, trigoneline and sucrose in
coffee beans.
Acknowledgements
The authors primarily offer their deepest heart-felt thanks
and glory to Almighty GOD. The authors are also
acknowledge Wollega University and Ministry of Science
and Higher Education for fellowships and financial support.
Appendix
Appendix A: Chromatogram of 2 mg/L of Standard Caffeine
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17 13
Appendix B: Chromatogram of 4 mg/L of Standard Caffeine
Appendix C: Chromatogram of 8 mg/L of Standard Caffeine
Appendix D: Chromatogram of 16 mg/L of Standard Caffeine
14 Shibiru Eticha and Tesfa Bedassa: Determination of Caffeine in Coffee Samples by High Performance Liquid
Chromatography and Ultra Violet - Visible Spectrophotometry Methods from Wollega, Ethiopia
Appendix E: Chromatogram of Caffeine of Abe Dongoro Coffee Sample
Appendix F: Chromatogram of Caffeine of Gida Ayana Coffee Sample
Appendix G: Chromatogram of Caffeine of Sasiga Coffee Sample
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17 15
Appendix H: Chromatogram of Caffeine of Sibu Sire Coffee Sample
Appendix I: Chromatogram of Caffeine of Gida Ayana Coffee Sample (Spiked)
Appendix J: Chromatogram of Caffeine of Sasiga Coffee Sample (Duplicated)
16 Shibiru Eticha and Tesfa Bedassa: Determination of Caffeine in Coffee Samples by High Performance Liquid
Chromatography and Ultra Violet - Visible Spectrophotometry Methods from Wollega, Ethiopia
References
[1] Murthy, P. S. & Naidu, M. M. Sustainable management of coffee industry by-products and value addition. Resources, Conservation and Recycling, 2012, 66, 45–58.
[2] Yonas Belete, Bayetta Belachew & Chemeda Fininsa. Evaluation of bean qualities of indigenous Arabica Coffee genotypes across different environments. Journal of Plant Breeding and Crop Science, 2014, 6, 135-143.
[3] Mussattoa, I., Livia, M., Carneirob, P. A. & Teixeira, A. A study on chemical constituents and sugar extraction from spent coffee grounds. Carbohydrate Polymers, 2011, 83, 368–374.
[4] Gomez-Rui, J. N., David, S. L. & Jennifer, M. In vitro antioxidant activity of coffee compounds and their metabolites. Journal of Agriculture and Food Chemistry, 2007, 55, 6962-6969.
[5] Nuhu, A. Bioactive micronutrients in coffee: Recent analytical approaches for characterization and quantification. ISRN Nutrition. Retrieved from http://dx.doi.org/10.1155/2014/384230, 2014.
[6] Alonso-Salces, M., Francesca, S., Fabiano, R. & Berger, K. Botanical and geographical characterization of green coffee (Coffea arabica and Coffea canephora): Chemometric evaluation of phenolic and methylxanthine contents. Journal of Agriculture and Food Chemistry, 2009, 57, 4224–4235.
[7] Shishov, A., Volodina, N., Nechaeva, D., Gagarinova, S., & Bulatov, A. An automated homogeneous liquid-liquid microextraction based on deep eutectic solvent for the HPLC- UV determination of caffeine in beverages. Microchemical Journal, 2019, 144, 469-473.
[8] Vuong, V. & Roach, D. Caffeine in green tea. Its removal and isolation. Separation & Purification Reviews, 2014, 43, 155-174.
[9] Oba, S., Nagata, C., Nakamura, K., Fujii, K., Kawachi, T., Takatsuka, N. & Hiroyuki S. Consumption of coffee, green tea, oolong tea, black tea, chocolate snacks and the caffeine content in relation to risk of diabetes in Japanese men and women. British Journal of Nutrition, 2010, 103, 453–459.
[10] Bhupathiraju, S N., Pan, A., Vasanti, S., Manson, E., Willett, C., Dam, M. & Frank, B Caffeinated and caffeine-free beverages and risk of type 2 diabetes. American Journal of Clinical Nutrition, 2013, 97, 163–174.
[11] Mazzafera, P. & Silvarolla, M. Caffeine content variation in single green Arabica coffee seeds. Seed Science Research, 2010, 20, 163-167.
[12] Tello, J., Viguera, M. & Calvo, L. Extraction of caffeine from Robusta coffee (Coffea canephora vs Robusta) husks using supercritical carbon dioxide. The Journal of Supercritical Fluids, 2011, 59, 53–60.
[13] Nawrot, P., Jordan, S., Eastwood, J., Rotstein, J., Hugenholtz, A. & Feeley, M. Effects of caffeine on human health. Food Additives and Contaminants, 2003, 20, 1–30.
[14] Tsvetkova, B. G., Kostova, B. D., Rachev, D. R., Peikova, L. T. & Pencheva, I. P. HPLC assay and stability studies of
tablets containing paracetamol and caffeine. International Journal of Pharmaceutical Sciences Review and Research, 2013, 18, 138-142.
[15] Rodrigues, N. P. & Bragagnolo, N. Identification and quantification of bioactive compounds in coffee brews by HPLC–DAD–MSn. Journal of Food Composition and Analysis, 2013, 32, 105–115.
[16] Magalhaes, L. M., Sandia, M., Marcela, S., Joa, A. L. & Pascoa, N. M. Rapid assessment of bioactive phenolics and methylxanthines in spent coffee grounds by FT-NIRspectroscopy. Talanta, 2016, 143, 460–467.
[17] Vichare, V., Mujgond, P., Tambe, V. & Dhole, S. N. Simultaneous spectrophotometric determination of paracetamol and caffeine in tablet formulation. International Journal of PharmTech Research, 2010, 2, 2512-2516.
[18] Tautua, A. W., Bamidele, M. & Diepreye, E. R. Ultra-violet spectrophotometric determination of caffeine in soft and energy drinks available in Yenagoa, Nigeria. Advance Journal of Food Science and Technology, 2014, 6, 155-158.
[19] Rotko, T. K. & Bęczkowska, I. Nafion covered lead film electrode for the voltammetric determination of caffeine in beverage samples and pharmaceutical formulations. Food Chemistry, 2015, 172, 24-29.
[20] Maidon, B., Atikah, O. & Hermen, S. Study of various solvents for caffeine determination using UV-Vis Spectrophotometeric method. Journal of Applied Sciences Research, 2012, 8, 2439-2442.
[21] Navarra, G., Moschetti, M., Guarrasi, V., Mangione, M. R., Militello, V. & Leone. M. Simultaneous determination of caffeine and chlorogenic acids in green coffee by UV-Vis Spectroscopy. Journal of chemistry. Retrieved from https://doi.org/10.1155/2017/6435086, 2017.
[22] Mulu Hagos, Mesfin Redi-Abshiro, Bhagwan Singh Chandravanshi, Estifanos Ele Ahmed M. Mohammed & Hassen Mamo. The correlation between caffeine contents of green coffee beans and altitudes of the coffee plants grown in South West Ethiopia. Bulletin Chemical Society of Ethiopia, 2018, 32, 13-25.
[23] Abebe Belay, Kassahun Ture, Mesfin Redi & Araya Asfaw. Measurement of caffeine in coffee beans with UV-Vis spectrophotometry. Food Chemistry, 2008, 108, 310–315.
[24] Shrivastava, A. & Gupta, V. B. Methods for the determination of limit of detection and limit of quantitation of the Analytical methods. Chronicles of Young Scientists, 2011, 2, 21-25.
[25] Hecimovic, I., Cvitanovic, B. A., Horzic, D. & Komes. D. Comparative study of polyphenols and caffeine in different coffee varieties affected by the degree of roasting. Food Chemistry, 2011, 129, 991–1000.
[26] Hagos Yisak, Mesfin Redi-Abshiro & Bhagwan Singh Chandravanshi. Selective determination of caffeine and trigonelline in aqueous extract of green coffee beans by FT-MIR-ATR spectroscopy. Vibrational Spectroscopy, 2018, 97, 33–38.
[27] Gichimu, B. M., Gichuru, E. K., Mamati, G. & Nyende, A. B. Biochemical composition within Coffea arabica cv. Ruiru 11 and its relationship with cup quality. Journal of Food Research, 2014, 3, 31–44.
International Journal of Biochemistry, Biophysics & Molecular Biology 2020; 5(1): 8-17 17
[28] Sahar, A., Maryam, S. & Parisa, Z. Comparative Study of Mineral Elements and Caffeine in Imported Coffee Varieties Affected by the Degree of Roasting by HPLC Analysis. Journal of Chemical and Pharmaceutical Research, 2016, 8, 111-116.
[29] Belete Tewabe Gebeyehu & Solomon Libsu Bikila. Determination of caffeine content and antioxidant activity of coffee. American Journal of Applied Chemistry, 2015, 3, 69-76.
[30] Ephrem Demissie, Girma Woyessa & Arayaselassie Abebe. UV-Vis spectrophotometery determination of caffeine in green coffee beans from Hararghe, Ethiopia, using Beer-Lambert’s law and integrated absorption coefficient techniques. Scientific Study & Research Chemistry &
Chemical Engineering, Biotechnology, Food Industry, 2016, 17, 109-123.
[31] Maria, B. S., Paulo, M. & Marinez, A. L. Caffeine content of Ethiopian Coffea arabica beans. Genetics and Molecular Biology, 2000, 23, 213-215.
[32] Legesse Adane, Mesfine Shiferaw & Israel Alemayehu. Determination of Caffeine Content of Bale Coffee Using HPLC Analysis. Food Science and Quality Management, 2018, 73, 23-32.
[33] Dessalegn, Y., Labuscagne, M. T., Osthoff, G. & Herselman, L. Variation of green bean caffeine, chlorogenic acids, sucrose and trigolline contents among Ethiopian Arabica coffee accessions. Ethiopian Journal of Science, 2008, 30, 77–82.